Project Summary/Abstract Lung cancer is the leading cause of cancer-related deaths in the United States. While significant advances have been made in understanding the molecular aberrations that drive non-small cell lung cancer (NSCLC), driver mutations for approximately 50% of NSCLC patients are unknown. Identification and characterization of oncogenic driver mutations will be important in expanding the benefits of precision medicine to a broader patient population. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that controls multiple cellular processes dysregulated in cancer including cell growth, survival, metabolism, and cytoskeletal reorganization. mTOR functions in two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2. The current paradigm is that mTORC1 and mTORC2 are comprised of common (mTOR, mLST8) and unique components (Raptor or Rictor/Sin1, respectively). Amplification of RICTOR, a component of mTORC2, was recently identified as a driver and sole actionable target in 11% of NSCLC lung cancer patients. Additionally, other known drivers of NSCLC including loss of PTEN or activating mutations in PIK3CA are expected to increase reliance of tumor cells on mTORC2 signaling for survival. Unfortunately, while mTORC1 specific inhibitors and dual mTOR kinase inhibitors exist, an mTORC2 specific inhibitor is not yet available. Preliminary data presented in this proposal shows that genetic targeting of mLST8, a shared component of both mTOR complexes, specifically inhibits the activity of mTORC2 while leaving mTORC1 function intact, suggesting mLST8 could potentially be used as a target for mTORC2 specific inhibition. This proposal will (1) investigate the mechanism by which loss of mLST8 specifically inhibits mTORC2 function and (2) investigate the effects of mLST8 loss-of-function (LOF) on lung tumorigenesis. In order to understand the role of mLST8 in mTORC2 specific function, mLST8 will be mutated at its expected mTOR-binding residues, and downstream signaling and kinase function of mTORC2 will be tested using immunoblotting and kinase assays, respectively. The effects of these mutations on cell viability of mTORC2-dependent lung cancer cell lines will also be evaluated using MTT assays and a xenograft model. The efficacy of mLST8 as a target for mTORC2 specific inhibition will be tested in vivo using CRISPR/Cas9 to model mTORC2-dependent NSCLC, which will be monitored for tumor burden, cell proliferation, and apoptosis. Additionally, mTORC2 LOF and MEK inhibitor combination therapy will be tested in a murine KRAS-mutant model of NSCLC, for which there is currently no targeted therapy. The success of this project will have far-reaching significance by advancing our understanding of the ubiquitous mTOR signaling pathway, while also increasing the translational potential of new therapies for NSCLC by identifying a new target for mTORC2 specific inhibition.